The role of adenine nucleotide binding sites and of the Pi binding site on beef heart mitochondrial ATPase will be explored. We shall study the interaction of the Pi site with the adenine nucleotide sites by measuring equilibrium binding of Pi to samples of the enzyme which contain varying amounts of adenine nucleotides. Such samples of the enzyme will be prepared by first removing all of the nucleotides on columns of sephadex equilibrated with 50% glycerol and then allowing adenine nucleotides to rebind to the enzyme under controlled conditions which will result in predictable occupancy of sites. The function of the various sites will be further pursued through chemical modification followed by ATPase and coupling factor activities of the resulting preparation.